Nutritional modulation of the host immune response to infection has been investigated in the critically ill with varied results, predominantly due to the heterogeneity of disease and differences in the composition of the dietary treatment. Understanding the function of individual compounds with respect to immunity would therefore be essential in evaluating the efficacy of dietary formulations. Dietary niacin undergoes several redox reactions resulting in the production of nicotinamide (NAM). Enzymes in the cytosol, mitochondria, and nucleus catalyze NAM transformation to NAD where the molecule is used in NAD(P)H redox reactions or consumed as a substrate by poly(ADP-ribose) polymerases (PARPs), CD38/CD157 ectoenzymes and sirtuins. Research has indicated that the disparate functions of myeloid cells may be linked to altered cell metabolism such that a pro-inflammatory (M1) macrophage utilizes anaerobic metabolism whereas an alternatively activated (M2) macrophage, involved in wound repair and tissue homeostasis, utilizes oxidative phosphorylation (OXPHOS) metabolism. The transcription factor HIF-1alpha is pivotal to anaerobic metabolism and a functional component in macrophage migration, phagocytosis, and antigen presentation. Macrophages isolated from HIF-1alpha deficient mice exhibit impaired clearance of gram positive and negative bacteria whereas pharmacological augmentation of HIF-1alpha boosts the response. Additional in vitro analysis of human monocyte derived macrophages (HMDMs) revealed that NAM antagonizes LPS-induced oxidative stress. These antioxidant properties of NAM decreased LPS-induced M1 pro-inflammatory cytokine production (tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1beta) and increased components of an M2 phenotype (CD200 receptor and C-type mannose receptor 1 expression). In the field of immuno-oncology, blockade of the programmed death 1 (PD-1) axis is being examined as a means to induce anti-tumor immunity. Antibodies to PD-1 or PD-L1 are expected to block T cell PD-1 binding with its ligand (PD-L1, PD-L2) on antigen presentation cells or tumor cells and thereby antagonize T cell PD-1 cell signals and promote anti-tumor immunity. The cell surface receptor, CD38, is an activation marker that mediates the catabolization of extracelluar NAD into intracellular NAM and cyclic ADP-ribose involved in Ca2+ cell signals. This receptor is also a recently identified biomarker and target in tumors resistant to anti-PD-L1/PD-1 therapy. In response to lipopolysaccharide (LPS), macrophages increase their expression of PD-L1 and CD38 on the cell surface, suggesting that NAM may also modify these responses. To begin to assess the functions of LPS and NAM with respect to metabolism, we examined if LPS could induce HIF-1alpha protein compared to a characterized hypoxia mimetic, DFOM, by immunoblot. Circulating monocytes from healthy volunteers were differentiated into HMDMs with 30 ng/ml M-CSF for 7 days. HMDMs (4x105 cells/ml) were incubated with 100 micro M DFOM or 10 ng/ml LPS for 24 hours. Cells were solubilized and immunoblots were probed for HIF-1alpha or actin. We identified a 120 kDa band associated with HIF-1alpha production that is up-regulated by DFOM and LPS compared to the control. To assess if NAM is able modulate the production of HIF-1alpha, we treated HMDMs with 10 ng/ml LPS in the absence or presence of increasing concentrations of NAM for 24 hours. We showed decreased production of HIF-1alpha in response to increasing concentrations of NAM compared to the LPS control. To assess NAM-induced transcriptional responses, the endothelial cell line, EAhy926, was transfected with reporter plasmids encoding hypoxia response elements (HRE) or NF-kB response elements (NRE). HRE and NRE transcriptional responses and HIF-1alpha production were inhibited by NAM at 5h and 24h respectively in these cells. In examining characterized downstream responses of HIF-1alpha in HMDMs, cell surface markers involved in antigen presentation, myeloid suppression, and activation were assessed. We identify increased expression of CD80, CD40, MHCI, PD-L1, and CD38 in the presence of LPS that is significantly reduced by NAM. Because NAD is produced from either dietary tryptophan or NAM, expression of the respective rate limiting enzymes, IDO and NAMPT, were assessed in HMDMs. The LPS-induced production of IDO was significantly reduced by NAM. LPS-induced NAMPT or the associated secondary enzyme the NAM pathway, NMNAT1, were not affected by NAM. In assessing the potential HMDM production of NAD in response to NAM treatment, the ratio of NAD to NADH was measured utilizing a luciferin reductase substrate. This assay identified an increase in the NAD/NADH ratio in response to NAM. Utilizing a NAMPT inhibitor reduced the NAD/NADH ratio in the presence of NAM but did not rescue the expression of HIF-1alpha in NAM treatments. Additional experiments with a SIRT activator or inhibitor, which requires NAD for activity, also did not significantly alter the NAM-induced HIF-1alpha response. To understand if NAM has redox potential, NAM treatment was compared to a characterized antioxidant, N-acetyl cysteine (NAC). NAC similarly reduced the LPS-induced production of HIF-1alpha and expression of CD40, PD-L1, and CD38. However, NAC inhibited the production of LPS-induced reactive oxygen species (ROS) in a bioluminescent assay whereas NAM did not. This suggests that NAM operates by an additional mechanism. In examining the prolyl hydroxylases (PHDs), we identified that PHD-2 is significantly upregulated by NAM. PHD2 is the main hydroxylase involved in the degradation of HIF-1alpha. This protein is significantly upregulated by NAM. The regulation of HIF-1alpha by NAM suggestively involves PHD2. The use of a PHD2 inhibitor to block NAM induced effects is being assessed. Further evaluation of PHD2 function continues to be explored. In summary, our preliminary studies have shown that in vitro treatment of human macrophages with NAM is able to reduce the expression of LPS-induced HIF-1alpha, MHCI, CD40, CD80, PD-L1 and CD38. We have identified the regulation HIF-1alpha and NF-kB transcriptional responses by NAM. The production of NAD in macrophages is enhanced by NAM, possibly by steady activation of enzymes, NAMPT and NMNAT1. Levels of NAD do not appear to directly influence SIRT1 activity. Although the antioxidant, NAC, can exhibit similar responses compared to NAM, the production of ROS is not blocked by NAM. A mechanism by which NAM functions involves the upregulation of PHD2. Continued exploration of NAM in LPS-induced hypoxic cell signals will provide insight into bacterial inflammation, hypoxia, and the immune responses associated with PD-1/PD-L1 and CD38.